Search

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRH OB ) of adult mice can mediate social recognition. Specifically, we show that GnRH OB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRH OB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRH OB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRH OB neurons' actions on male mounting behavior. Taken together, these results establish GnRH OB neurons as regulating fertility, sex recognition and mating in male mice., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The fossorial water vole, Arvicola scherman, is an herbivorous rodent that causes significant agricultural damages. The application of cairomones and alarm pheromones emerges as a promising sustainable method to improve its integrated management. These chemical signals would induce stress responses that could interfere with the species regular reproductive cycles and induce aversive reactions, steering them away from farmlands and meadows. However, there is a paucity of information regarding the water vole vomeronasal system, both in its morphological foundations and its functionality, making it imperative to understand the same for the application of chemical communication in pest control. This study fills the existing gaps in knowledge through a morphological and immunohistochemical analysis of the fossorial water vole vomeronasal organ. The study is primarily microscopic, employing two approaches: histological, using serial sections stained with various dyes (hematoxylin-eosin, Periodic acid-Schiff, Alcian blue, Nissl), and immunohistochemical, applying various markers that provide morphofunctional and structural information. These procedures have confirmed the presence of a functional vomeronasal system in fossorial water voles, characterized by a high degree of differentiation and a significant expression of cellular markers indicative of active chemical communication in this species., (© 2023 The Authors. The Anatomical Record published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Wolves, akin to their fellow canids, extensively employ chemical signals for various aspects of communication, including territory maintenance, reproductive synchronisation and social hierarchy signalling. Pheromone-mediated chemical communication operates unconsciously among individuals, serving as an innate sensory modality that regulates both their physiology and behaviour. Despite its crucial role in the life of the wolf, there is a lacuna in comprehensive research on the neuroanatomical and physiological underpinnings of chemical communication within this species. This study investigates the vomeronasal system (VNS) of the Iberian wolf, simultaneously probing potential alterations brought about by dog domestication. Our findings demonstrate the presence of a fully functional VNS, vital for pheromone-mediated communication, in the Iberian wolf. While macroscopic similarities between the VNS of the wolf and the domestic dog are discernible, notable microscopic differences emerge. These distinctions include the presence of neuronal clusters associated with the sensory epithelium of the vomeronasal organ (VNO) and a heightened degree of differentiation of the accessory olfactory bulb (AOB). Immunohistochemical analyses reveal the expression of the two primary families of vomeronasal receptors (V1R and V2R) within the VNO. However, only the V1R family is expressed in the AOB. These findings not only yield profound insights into the VNS of the wolf but also hint at how domestication might have altered neural configurations that underpin species-specific behaviours. This understanding holds implications for the development of innovative strategies, such as the application of semiochemicals for wolf population management, aligning with contemporary conservation goals., (© 2024 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

In most mammals, conspecific chemosensory communication relies on semiochemical release within complex bodily secretions and subsequent stimulus detection by the vomeronasal organ (VNO). Urine, a rich source of ethologically relevant chemosignals, conveys detailed information about sex, social hierarchy, health, and reproductive state, which becomes accessible to a conspecific via vomeronasal sampling. So far, however, numerous aspects of social chemosignaling along the vomeronasal pathway remain unclear. Moreover, since virtually all research on vomeronasal physiology is based on secretions derived from inbred laboratory mice, it remains uncertain whether such stimuli provide a true representation of potentially more relevant cues found in the wild. Here, we combine a robust low-noise VNO activity assay with comparative molecular profiling of sex- and strain-specific mouse urine samples from two inbred laboratory strains as well as from wild mice. With comprehensive molecular portraits of these secretions, VNO activity analysis now enables us to (i) assess whether and, if so, how much sex/strain-selective 'raw' chemical information in urine is accessible via vomeronasal sampling; (ii) identify which chemicals exhibit sufficient discriminatory power to signal an animal's sex, strain, or both; (iii) determine the extent to which wild mouse secretions are unique; and (iv) analyze whether vomeronasal response profiles differ between strains. We report both sex- and, in particular, strain-selective VNO representations of chemical information. Within the urinary 'secretome', both volatile compounds and proteins exhibit sufficient discriminative power to provide sex- and strain-specific molecular fingerprints. While total protein amount is substantially enriched in male urine, females secrete a larger variety at overall comparatively low concentrations. Surprisingly, the molecular spectrum of wild mouse urine does not dramatically exceed that of inbred strains. Finally, vomeronasal response profiles differ between C57BL/6 and BALB/c animals, with particularly disparate representations of female semiochemicals., Competing Interests: MN, MN, RB, DF, AL, RS, PS, YB, MS No competing interests declared, (© 2023, Nagel et al.)

In mammals, especially rodents, social behaviours, such as parenting, territoriality or mate attraction, are largely based on olfactory communication through chemosignals. These behaviours are mediated by species-specific chemosignals, including small organic molecules and proteins that are secreted in the urine or in various fluids from exocrine glands. Chemosignal detection is mainly ensured by olfactory neurons in two specific sensory organs, the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). This study aimed to characterise the olfactory communication in the fossorial ecotype of the water voles, Arvicola terrestris. We first measured the olfactory investigation of urine and lateral scent gland secretions from conspecifics. Our results showed that water voles can discriminate the sex of conspecifics based on the smell of urine, and that urinary male odour is attractive for female voles. Then, we demonstrated the ability of the VNO and MOE to detect volatile organic compounds (VOCs) found in water vole secretions using live-cell calcium imaging in dissociated cells. Finally, we evaluated the attractiveness of two mixtures of VOCs from urine or lateral scent glands in the field during a cyclical outbreak of vole populations., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pheromones are specialized chemical messengers used for inter-individual communication within the same species, playing crucial roles in modulating behaviors and physiological states. The detection mechanisms of these signals at the peripheral organ and their transduction to the brain have been unclear. However, recent identification of pheromone molecules, their corresponding receptors, and advancements in neuroscientific technology have started to elucidate these processes. In mammals, the detection and interpretation of pheromone signals are primarily attributed to the vomeronasal system, which is a specialized olfactory apparatus predominantly dedicated to decoding socio-chemical cues. In this mini-review, we aim to delineate the vomeronasal signal transduction pathway initiated by specific vomeronasal receptor-ligand interactions in mice. First, we catalog the previously identified pheromone ligands and their corresponding receptor pairs, providing a foundational understanding of the specificity inherent in pheromonal communication. Subsequently, we examine the neural circuits involved in processing each pheromone signal. We focus on the anatomical pathways, the sexually dimorphic and physiological state-dependent aspects of signal transduction, and the neural coding strategies underlying behavioral responses to pheromonal cues. These insights provide further critical questions regarding the development of innate circuit formation and plasticity within these circuits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Murata, Itakura and Touhara.)

The physiological performance of any sensory organ is determined by its anatomy and physical properties. Consequently, complex sensory structures with elaborate features have evolved to optimize stimulus detection. Understanding these structures and their physical nature forms the basis for mechanistic insights into sensory function. Despite its crucial role as a sensor for pheromones and other behaviorally instructive chemical cues, the vomeronasal organ (VNO) remains a poorly characterized mammalian sensory structure. Fundamental principles of its physico-mechanical function, including basic aspects of stimulus sampling, remain poorly explored. Here, we revisit the classical vasomotor pump hypothesis of vomeronasal stimulus uptake. Using advanced anatomical, histological, and physiological methods, we demonstrate that large parts of the lateral mouse VNO are composed of smooth muscle. Vomeronasal smooth muscle tissue comprises two subsets of fibers with distinct topography, structure, excitation-contraction coupling, and, ultimately, contractile properties. Specifically, contractions of a large population of noradrenaline-sensitive cells mediate both transverse and longitudinal lumen expansion, whereas cholinergic stimulation targets an adluminal group of smooth muscle fibers. The latter run parallel to the VNO's rostro-caudal axis and are ideally situated to mediate antagonistic longitudinal constriction of the lumen. This newly discovered arrangement implies a novel mode of function. Single-cell transcriptomics and pharmacological profiling reveal the receptor subtypes involved. Finally, 2D/3D tomography provides non-invasive insight into the intact VNO's anatomy and mechanics, enables measurement of luminal fluid volume, and allows an assessment of relative volume change upon noradrenergic stimulation. Together, we propose a revised conceptual framework for mouse vomeronasal pumping and, thus, stimulus sampling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

While we understand how the five main sensory organs enable and facilitate stimulus detection, little is known about how the vomeronasal organ enables pheromone sensation. A new study finds specialized muscles poised to coordinate stimulus delivery, dynamics, and arousal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity., (© 2024 Wiley Periodicals LLC.)

Background: Rodents utilize chemical cues to recognize and avoid other conspecifics infected with pathogens. Infection with pathogens and acute inflammation alter the repertoire and signature of olfactory stimuli emitted by a sick individual. These cues are recognized by healthy conspecifics via the vomeronasal or accessory olfactory system, triggering an innate form of avoidance behavior. However, the molecular identity of the sensory neurons and the higher neural circuits involved in the detection of sick conspecifics remain poorly understood., Results: We employed mice that are in an acute state of inflammation induced by systemic administration of lipopolysaccharide (LPS). Through conditional knockout of the G-protein Gαi2 and deletion of other key sensory transduction molecules (Trpc2 and a cluster of 16 vomeronasal type 1 receptors), in combination with behavioral testing, subcellular Ca 2+ imaging, and pS6 and c-Fos neuronal activity mapping in freely behaving mice, we show that the Gαi2 + vomeronasal subsystem is required for the detection and avoidance of LPS-treated mice. The active components underlying this avoidance are contained in urine whereas feces extract and two selected bile acids, although detected in a Gαi2-dependent manner, failed to evoke avoidance behavior. Our analyses of dendritic Ca 2+ responses in vomeronasal sensory neurons provide insight into the discrimination capabilities of these neurons for urine fractions from LPS-treated mice, and how this discrimination depends on Gαi2. We observed Gαi2-dependent stimulation of multiple brain areas including medial amygdala, ventromedial hypothalamus, and periaqueductal grey. We also identified the lateral habenula, a brain region implicated in negative reward prediction in aversive learning, as a previously unknown target involved in these tasks., Conclusions: Our physiological and behavioral analyses indicate that the sensing and avoidance of LPS-treated sick conspecifics depend on the Gαi2 vomeronasal subsystem. Our observations point to a central role of brain circuits downstream of the olfactory periphery and in the lateral habenula in the detection and avoidance of sick conspecifics, providing new insights into the neural substrates and circuit logic of the sensing of inflammation in mice., (© 2023. The Author(s).)

Introduction: Contactin-6 (CNTN6), also known as NB-3, is a neural recognition molecule and a member of the contactin subgroup of the immunoglobulin superfamily. Gene encoding CNTN6 is expressed in many regions of the neural system, including the accessory olfactory bulb (AOB) in mice. We aim to determine the effect of CNTN6 deficiency on the function of the accessory olfactory system (AOS)., Methods: We examined the effect of CNTN6 deficiency on the reproductive behavior of male mice through behavioral experiments such as urine sniffing and mate preference tests. Staining and electron microscopy were used to observe the gross structure and the circuitry activity of the AOS., Results: Cntn6 is highly expressed in the vomeronasal organ (VNO) and the AOB, and sparsely expressed in the medial amygdala (MeA) and the medial preoptic area (MPOA), which receive direct and/or indirect projections from the AOB. Behavioral tests to examine reproductive function in mice, which is mostly controlled by the AOS, revealed that Cntn6 -/- adult male mice showed less interest and reduced mating attempts toward estrous female mice in comparison with their Cntn6 +/+ littermates. Although Cntn6 -/- adult male mice displayed no obvious changes in the gross structure of the VNO or AOB, we observed the increased activation of granule cells in the AOB and the lower activation of neurons in the MeA and the MPOA as compared with Cntn6 +/+ adult male mice. Moreover, there were an increased number of synapses between mitral cells and granule cells in the AOB of Cntn6 -/- adult male mice as compared with wild-type controls., Conclusion: These results indicate that CNTN6 deficiency affects the reproductive behavior of male mice, suggesting that CNTN6 participated in normal function of the AOS and its ablation was involved in synapse formation between mitral and granule cells in the AOB, rather than affecting the gross structure of the AOS., (© 2023 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

The pheromonal information received by the vomeronasal system plays a crucial role in regulating social behaviors such as aggression in mice. Despite accumulating knowledge of the brain regions involved in aggression, the specific vomeronasal receptors and the exact neural circuits responsible for pheromone-mediated aggression remain unknown. Here, we identified one murine vomeronasal receptor, Vmn2r53, that is activated by urine from males of various strains and is responsible for evoking intermale aggression. We prepared a purified pheromonal fraction and Vmn2r53 knockout mice and applied genetic tools for neuronal activity recording, manipulation, and circuit tracing to decipher the neural mechanisms underlying Vmn2r53-mediated aggression. We found that Vmn2r53-mediated aggression is regulated by specific neuronal populations in the ventral premammillary nucleus and the ventromedial hypothalamic nucleus. Together, our results shed light on the hypothalamic regulation of male aggression mediated by a single vomeronasal receptor., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adaptation plays an important role in sensory systems as it dynamically modifies sensitivity to allow the detection of stimulus changes. The vomeronasal system controls many social behaviors in most mammals by detecting pheromones released by conspecifics. Stimuli activate a transduction cascade in vomeronasal neurons that leads to spiking activity. Whether and how these neurons adapt to stimuli is still debated and largely unknown. Here, we measured short-term adaptation performing current-clamp whole-cell recordings by using diluted urine as a stimulus, as it contains many pheromones. We measured spike frequency adaptation in response to repeated identical stimuli of 2-10 s duration that was dependent on the time interval between stimuli. Responses to paired current steps, bypassing the signal transduction cascade, also showed spike frequency adaptation. We found that voltage-gated Na + channels in VSNs undergo slow inactivation processes. Furthermore, recovery from slow inactivation of voltage-gated Na + channels occurs in several seconds, a time scale similar to that measured during paired-pulse adaptation protocols, suggesting that it partially contributes to short-term spike frequency adaptation. We conclude that vomeronasal neurons do exhibit a time-dependent short-term spike frequency adaptation to repeated natural stimuli and that slow inactivation of Na + channels contributes to this form of adaptation. These findings not only increase our knowledge about adaptation in the vomeronasal system, but also raise the question of whether slow inactivation of Na + channels may play a role in other sensory systems., (Copyright © 2022 Sarno et al.)

Vomeronasal information is critical in mice for territorial behavior. Consequently, learning the territorial spatial structure should incorporate the vomeronasal signals indicating individual identity into the hippocampal cognitive map. In this work we show in mice that navigating a virtual environment induces synchronic activity, with causality in both directionalities, between the vomeronasal amygdala and the dorsal CA1 of the hippocampus in the theta frequency range. The detection of urine stimuli induces synaptic plasticity in the vomeronasal pathway and the dorsal hippocampus, even in animals with experimentally induced anosmia. In the dorsal hippocampus, this plasticity is associated with the overexpression of pAKT and pGSK3β. An amygdalo-entorhino-hippocampal circuit likely underlies this effect of pheromonal information on hippocampal learning. This circuit likely constitutes the neural substrate of territorial behavior in mice, and it allows the integration of social and spatial information., (© 2021. The Author(s).)

During lactation, adult female mice display aggressive responses toward male intruders, triggered by male-derived chemosensory signals. This aggressive behavior is not shown by pup-sensitized virgin females sharing pup care with dams. The genetic mechanisms underlying the switch from attraction to aggression are unknown. In this work, we investigate the differential gene expression in lactating females expressing maternal aggression compared to pup-sensitized virgin females in the medial amygdala (Me), a key neural structure integrating chemosensory and hormonal information. The results showed 197 genes upregulated in dams, including genes encoding hormones such as prolactin, growth hormone, or follicle-stimulating hormone, neuropeptides such as galanin, oxytocin, and pro-opiomelanocortin, and genes related to catecholaminergic and cholinergic neurotransmission. In contrast, 99 genes were downregulated in dams, among which we find those encoding for inhibins and transcription factors of the Fos and early growth response families. The gene set analysis revealed numerous Gene Ontology functional groups with higher expression in dams than in pup-sensitized virgin females, including those related with the regulation of the Jak/Stat cascade. Of note, a number of olfactory and vomeronasal receptor genes was expressed in the Me, although without differences between dams and virgins. For prolactin and growth hormone, a qPCR experiment comparing dams, pup-sensitized, and pup-naïve virgin females showed that dams expressed higher levels of both hormones than pup-naïve virgins, with pup-sensitized virgins showing intermediate levels. Altogether, the results show important gene expression changes in the Me, which may underlie some of the behavioral responses characterizing maternal behavior., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Communication between individuals is critical for species survival, reproduction, and expansion. Most terrestrial species, with the exception of humans who predominantly use vision and phonation to create their social network, rely on the detection and decoding of olfactory signals, which are widely known as pheromones. These chemosensory cues originate from bodily fluids, causing attractive or avoidance behaviors in subjects of the same species. Intraspecific pheromone signaling is then crucial to identify sex, social ranking, individuality, and health status, thus establishing hierarchies and finalizing the most efficient reproductive strategies. Indeed, all these features require fine tuning of the olfactory systems to detect molecules containing this information. To cope with this complexity of signals, tetrapods have developed dedicated olfactory subsystems that refer to distinct peripheral sensory detectors, called the main olfactory and the vomeronasal organ, and two minor structures, namely the septal organ of Masera and the Grueneberg ganglion. Among these, the vomeronasal organ plays the most remarkable role in pheromone coding by mediating several behavioral outcomes that are critical for species conservation and amplification. In rodents, this organ is organized into two segregated neuronal subsets that express different receptor families. To some extent, this dichotomic organization is preserved in higher projection areas of the central nervous system, suggesting, at first glance, distinct functions for these two neuronal pathways. Here, I will specifically focus on this issue and discuss the role of vomeronasal receptors in mediating important innate behavioral effects through the recognition of pheromones and other biological chemosignals.

The hypothalamus regulates innate social interactions, but how hypothalamic neurons transduce sex-related sensory signals emitted by conspecifics to trigger appropriate behaviors remains unclear. Here, we addressed this issue by identifying specific hypothalamic neurons required for sensing conspecific male cues relevant to inter-male aggression. By in vivo recording of neuronal activities in behaving mice, we showed that neurons expressing dopamine transporter (DAT + ) in the ventral premammillary nucleus (PMv) of the hypothalamus responded to male urine cues in a vomeronasal organ (VNO)-dependent manner in naive males. Retrograde trans-synaptic tracing further revealed a specific group of neurons in the bed nucleus of the stria terminalis (BNST) that convey male-relevant signals from VNO to PMv. Inhibition of PMv DAT+ neurons abolished the preference for male urine cues and reduced inter-male attacks, while activation of these neurons promoted urine marking and aggression. Thus, PMv DAT+ neurons exemplify a hypothalamic node that transforms sex-related chemo-signals into recognition and behaviors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Behaviors are shaped by hormones, which may act either by changing brain circuits or by modifying sensory detection of relevant cues. Pup-directed behaviors have been previously shown to change via action of hormones at the brain level. Here, we investigated hormonal control of pup-induced activity in the vomeronasal organ, an olfactory sensory structure involved in the detection of non-volatile chemosignals. Vomeronasal activity decreases as males switch from a pup-aggressive state to a non-aggressive parenting state, after they socially contact a female. RNA sequencing, qPCR, and in situ hybridization were used to identify expression, in the vomeronasal sensory epithelium, of candidate GPCR hormone receptors chosen by in silico analyses and educated guesses. After identifying that oxytocin and vasopressin receptors are expressed in the vomeronasal organ, we injected the corresponding hormones in mice and showed that oxytocin administration reduced both pup-induced vomeronasal activity and aggressive behavior. Conversely, injection of an oxytocin receptor antagonist in female-primed male animals, which normally exhibit reduced vomeronasal activity, significantly increased the number of active vomeronasal neurons. These data link oxytocin to the modulation of olfactory sensory activity, providing a possible mechanism for changes in male behavior after social experience with females.

The accessory olfactory bulb (AOB), the first neural circuit in the mouse accessory olfactory system, is critical for interpreting social chemosignals. Despite its importance, AOB information processing is poorly understood compared with the main olfactory bulb (MOB). Here, we sought to fill gaps in the understanding of AOB interneuron function. We used 2-photon GCaMP6f Ca 2+ imaging in an ex vivo preparation to study chemosensory tuning in AOB external granule cells (EGCs), interneurons hypothesized to broadly inhibit activity in excitatory mitral cells (MCs). In ex vivo preparations from mice of both sexes, we measured MC and EGC tuning to natural chemosignal blends and monomolecular ligands, finding that EGC tuning was sparser, not broader, than upstream MCs. Simultaneous electrophysiological recording and Ca 2+ imaging showed no differences in GCaMP6f-to-spiking relationships in these cell types during simulated sensory stimulation, suggesting that measured EGC sparseness was not due to cell type-dependent variability in GCaMP6f performance. Ex vivo patch-clamp recordings revealed that EGC subthreshold responsivity was far broader than indicated by GCaMP6f Ca 2+ imaging, and that monomolecular ligands rarely elicited EGC spiking. These results indicate that EGCs are selectively engaged by chemosensory blends, suggesting different roles for EGCs than analogous interneurons in the MOB. SIGNIFICANCE STATEMENT The mouse accessory olfactory system (AOS) interprets social chemosignals, but we poorly understand AOS information processing. Here, we investigate the functional properties of external granule cells (EGCs), a major class of interneurons in the accessory olfactory bulb (AOB). We hypothesized that EGCs, which are densely innervated by excitatory mitral cells (MCs), would show broad chemosensory tuning, suggesting a role in divisive normalization. Using ex vivo GCaMP6f imaging, we found that EGCs were instead more sparsely tuned than MCs. This was not due to weaker GCaMP6f signaling in EGCs than in MCs. Instead, we found that many MC-activating chemosignals caused only subthreshold EGC responses. This indicates a different role for AOB EGCs compared with analogous cells in the main olfactory bulb., (Copyright © 2020 Zhang and Meeks.)

We summarize literature from animal and human studies assessing sex differences in the ability of the main olfactory system to detect and process sex-specific olfactory signals ("pheromones") that control the expression of psychosexual functions in males and females. A case is made in non primate mammals for an obligatory role of pheromonal signaling via the main olfactory system (in addition to the vomeronasal-accessory olfactory system) in mate recognition and sexual arousal, with male-specific as well as female-specific pheromones subserving these functions in the opposite sex. Although the case for an obligatory role of pheromones in mate recognition and mating among old world primates, including humans, is weaker, we review the current literature assessing the role of putative human pheromones (eg, AND, EST, "copulin"), detected by the main olfactory system, in promoting mate choice and mating in men and women. Based on animal studies, we hypothesize that sexually dimorphic effects of putative human pheromones are mediated via main olfactory inputs to the medial amygdala which, in turn, transmits olfactory information to sites in the hypothalamus that regulate reproduction., (© 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

The colonial naked mole rat Heterocephalus glaber is a subterranean, eusocial rodent. The H. glaber vomeronasal organ neuroepithelium (VNE) displays little postnatal growth. However, the VNE remains neuronal in contrast to some mammals that possess nonfunctional vomeronasal organ remnants, for example, catarrhine primates and some bats. Here, we describe the vomeronasal organ (VNO) microanatomy in the naked mole rat and we make preliminary observations to determine if H. glaber shares its minimal postnatal VNE growth with other African mole rats. We also determine the immunoreactivity to the mitotic marker Ki67, growth-associated protein 43 (GAP43), and olfactory marker protein (OMP) in six adult and three subadult H. glaber individuals. VNE volume measurements on a small sample of Cryptomys hottentotus and Fukomys damarensis indicate that the VNE of those African mole rat species are also likely to be growth-deficient. Ki67(+) cells show that the sensory epithelium is mitotically active. GAP43 labelling indicates neurogenesis and OMP(+) cells are present though less numerous compared to GAP43(+) cells. In this respect, the VNO of H. glaber does not appear vestigial. The African mole rat VNE may be unusually variable, perhaps reflecting reduced selection pressure on the vomeronasal system. If so, African mole rats may provide a useful genetic model for understanding the morphological variability observed in the mammalian VNO. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 303:318-329, 2020. © 2019 American Association for Anatomy., (© 2019 Wiley Periodicals, Inc.)

Pheromone detection by the vomeronasal organ (VNO) mediates important social behaviors across different species, including aggression and sexual behavior. However, the relationship between vomeronasal function and social hierarchy has not been analyzed reliably. We evaluated the role of pheromone detection by receptors expressed in the apical layer of the VNO such as vomeronasal type 1 receptors (V1R) in dominance behavior by using a conditional knockout mouse for G protein subunit Gαi2, which is essential for V1R signaling. We used the tube test as a model to analyze the within-a-cage hierarchy in male mice, but also as a paradigm of novel territorial competition in animals from different cages. In absence of prior social experience, Gαi2 deletion promotes winning a novel social competition with an unfamiliar control mouse but had no effect on an established hierarchy in cages with mixed genotypes, both Gαi2 -/- and controls. To further dissect social behavior of Gαi2 -/- mice, we performed a 3-chamber sociability assay and found that mutants had a slightly altered social investigation. Finally, gene expression analysis in the medial prefrontal cortex (mPFC) for a subset of genes previously linked to social status revealed no differences between group-housed Gαi2 -/- and controls. Our results reveal a direct influence of pheromone detection on territorial dominance, indicating that olfactory communication involving apical VNO receptors like V1R is important for the outcome of an initial social competition between two unfamiliar male mice, whereas final social status acquired within a cage remains unaffected. These results support the idea that previous social context is relevant for the development of social hierarchy of a group. Overall, our data identify two context-dependent forms of dominance, acute and chronic, and that pheromone signaling through V1R receptors is involved in the first stages of a social competition but in the long term is not predictive for high social ranks on a hierarchy.

During mammalian development, gonadotropin-releasing-hormone-1 neurons (GnRH-1ns) migrate from the developing vomeronasal organ (VNO) into the brain asserting control of pubertal onset and fertility. Recent data suggest that correct development of the olfactory ensheathing cells (OEC) is imperative for normal GnRH-1 neuronal migration. However, the full ensemble of molecular pathways that regulate OEC development remains to be fully deciphered. Loss-of-function of the transcription factor Gli3 is known to disrupt olfactory development, however, if Gli3 plays a role in GnRH-1 neuronal development is unclear. By analyzing Gli3 extra-toe mutants (Gli3 Xt/Xt ), we found that Gli3 loss-of-function compromises the onset of achaete-scute family bHLH transcription factor 1 (Ascl-1) + vomeronasal progenitors and the formation of OEC in the nasal mucosa. Surprisingly, GnRH-1 neurogenesis was intact in Gli3 Xt/Xt mice but they displayed significant defects in GnRH-1 neuronal migration. In contrast, Ascl-1 null mutants showed reduced neurogenesis for both vomeronasal and GnRH-1ns but less severe defects in OEC development. These observations suggest that Gli3 is critical for OEC development in the nasal mucosa and subsequent GnRH-1 neuronal migration. However, the nonoverlapping phenotypes between Ascl-1 and Gli3 mutants indicate that Ascl-1, while crucial for GnRH-1 neurogenesis, is not required for normal OEC development. Because Kallmann syndrome (KS) is characterized by abnormal GnRH-1ns migration, we examined whole-exome sequencing data from KS subjects. We identified and validated a GLI3 loss-of-function variant in a KS individual. These findings provide new insights into GnRH-1 and OECs development and demonstrate that human GLI3 mutations contribute to KS etiology. SIGNIFICANCE STATEMENT The transcription factor Gli3 is necessary for correct development of the olfactory system. However, if Gli3 plays a role in controlling GnRH-1 neuronal development has not been addressed. We found that Gli3 loss-of-function compromises the onset of Ascl-1 + vomeronasal progenitors, formation of olfactory ensheathing cells in the nasal mucosa, and impairs GnRH-1 neuronal migration to the brain. By analyzing Ascl-1 null mutants we dissociated the neurogenic defects observed in Gli3 mutants from lack of olfactory ensheathing cells in the nasal mucosa, moreover, we discovered that Ascl-1 is necessary for GnRH-1 ontogeny. Analyzing human whole-exome sequencing data, we identified a GLI3 loss-of-function variant in a KS individual. Our data suggest that GLI3 is a candidate gene contributing to KS etiology., (Copyright © 2020 Taroc et al.)

The vomeronasal system (VNS) serves crucial functions for detecting olfactory clues often related to social and sexual behaviour. Intriguingly, two of the main components of the VNS, the vomeronasal organ (VNO) and the accessory olfactory bulb, are regressed in aquatic mammals, several bats and primates, likely due to adaptations to different ecological niches. To detect genomic changes that are associated with the convergent reduction of the VNS, we performed the first systematic screen for convergently inactivated protein-coding genes associated with convergent VNS reduction, considering 106 mammalian genomes. Extending previous studies, our results support that Trpc2, a cation channel that is important for calcium signalling in the VNO, is a predictive molecular marker for the presence of a VNS. Our screen also detected the convergent inactivation of the calcium-binding protein S100z, the aldehyde oxidase Aox2 that is involved in odorant degradation, and the uncharacterized Mslnl gene that is expressed in the VNO and olfactory epithelium. Furthermore, we found that Trpc2 and S100z or Aox2 are also inactivated in otters and Phocid seals for which no morphological data about the VNS are available yet. This predicts a VNS reduction in these semi-aquatic mammals. By examining the genomes of 115 species in total, our study provides a detailed picture of how the convergent reduction of the VNS coincides with gene inactivation in placental mammals. These inactivated genes provide experimental targets for studying the evolution and biological significance of the olfactory system under different environmental conditions., (© 2019 John Wiley & Sons Ltd.)

Exocrine gland-secreting peptide 22 (ESP22) is a 10-kDa protein secreted in tears of juvenile mice. ESP22 inhibits sexual behaviors in adults, leading to a reduction in reproduction rate. We herein identified the 24 amino acid sequence within ESP22 that was essential for exhibiting sexual rejection activity. This synthesizable peptide can be useful for controlling mouse overpopulation.

The parallel processing of chemical signals by the main olfactory system and the vomeronasal system has been known to control animal behavior. The physiological significance of peripheral parallel pathways consisting of olfactory sensory neurons and vomeronasal sensory neurons is not well understood. Here, we show complementary characteristics of the information transfer of the olfactory sensory neurons and vomeronasal sensory neurons. A difference in excitability between the sensory neurons was revealed by patch-clamp experiments. The olfactory and vomeronasal sensory neurons showed phasic and tonic firing, respectively. Intrinsic channel kinetics determining firing patterns was demonstrated by a Hodgkin-Huxley-style computation. Our estimation of the information carried by action potentials during one cycle of sinusoidal stimulation with variable durations revealed distinct characteristics of information transfer between the sensory neurons. Phasic firing of the olfactory sensory neurons was suitable to carry information about rapid changes in a shorter cycle (<200 ms). In contrast, tonic firing of the vomeronasal sensory neurons was able to convey information about smaller stimuli changing slowly with longer cycles (>500 ms). Thus, the parallel pathways of the two types of sensory neurons can convey information about a wide range of dynamic stimuli. A combination of complementary characteristics of olfactory information transfer may enhance the synergy of the interaction between the main olfactory system and the vomeronasal system., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Olfactory experience can alter the molecular and cellular composition of chemosensory neurons within the olfactory sensory epithelia of mice. We sought to investigate the scope of cellular and molecular changes within a mouse's olfactory system as a function of its exposure to complex and salient sets of odors: those emitted from members of the opposite sex. We housed mice either separated from members of the opposite sex (sex-separated) or together with members of the opposite sex (sex-combined) until six months of age, resulting in the generation of four cohorts of mice. From each mouse, the main olfactory epithelium (MOE), vomeronasal organ (VNO), and olfactory bulb (OB) were removed and RNA-extracted. A total of 36 RNA samples, representing three biological replicates per sex/condition/tissue combination, were analyzed for integrity and used to prepare RNA-seq libraries, which were subsequently analyzed via qPCR for the presence of tissue- or sex-specific markers. Libraries were paired-end sequenced to a depth of ~20 million fragments per replicate and the data were analyzed using the Tuxedo suite.

The characterization of the rabbit mammary pheromone, which is sensed by the main olfactory system, has made this species a unique model for the study of pheromonal communication in mammals. This discovery has brought attention to the global understanding of chemosensory communication in this species. Chemocommunication is mediated by two distinct organs located in the nasal cavity, the main olfactory epithelium and the vomeronasal organ (VNO). However, there is a lack of knowledge about the vomeronasal system in rabbits. To understand the role of this system, an exhaustive anatomical and histological study of the rabbit VNO was performed. The rabbit VNO was studied macroscopically by light microscopy, and by histochemical and immunohistochemical techniques. We employed specific histological staining techniques (periodic acid-Schiff, Alcian blue, Gallego's trichrome), confocal autofluorescence, histochemical labelling with the lectin Ulex europaeus agglutinin (UEA-I), and immunohistochemical studies of the expression of the Gαi2 and Gαo proteins and olfactory marker protein. The opening of the vomeronasal duct into the nasal cavity and its indirect communication with the oral cavity through a functional nasopalatine duct was demonstrated by classical dissection and microdissection. In a series of transverse histological sections, special attention was paid to the general distribution of the various soft-tissue components of this organ (duct, glands, connective tissue, blood vessels and nerves) and to the nature of the capsule of the organ. Among the main morphological features that distinguish the rabbit VNO, the presence of a double envelope, which is bony externally and cartilaginous internally, and highly developed venous sinuses stand out. This observation indicates the crucial role played in this species by the pumping mechanism that introduces chemical signals into the vomeronasal duct. The functional properties of the organ are also confirmed by the presence of a well-developed neuroepithelium and profuse glandular tissue that is positive for neutral mucopolysaccharides. The role of glycoconjugates was assessed by the identification of the α1-2 fucose glycan system in the neuroepithelium of the VNO employing UEA-I lectin. The pattern of labelling, which was concentrated around the commissures of the sensory epithelium and more diffuse in the central segments, is different from that found in most mammals studied. According to the expression of G-proteins, two pathways have been described in the VNOs of mammals: neuroreceptor cells expressing the Gαi2 protein (associated with vomeronasal receptor type 1); and cells expressing Gαo (associated with vomeronasal receptor type 2). The latter pathway is absent in most mammals studied. The expression of both G-protein families in the rabbit VNO places Lagomorpha together with rodents and insectivores in a small group of mammals belonging to the two-path model. These findings support the notion that the rabbit possesses a highly developed VNO, with many specific morphological features, which highlights the significance of chemocommunication in this species., (© 2018 Anatomical Society.)

Animals respond to sensory cues emitted by their conspecifics by initiating a repertoire of sex-specific social behaviors, such as mating, fighting, and parental care. These behavioral responses are thought to be largely driven by genetically pre-programmed circuits. However, they can also vary significantly according to the animal's previous social experience and physiological state, suggesting that the underlying circuits are shaped by adaptive modulatory changes. Modern tools of neuroscience have enabled the direct interrogation of circuits driving sex-specific behaviors and helped unravel key neural mechanisms underlying their function and modulation. In particular, recent work has shown how individual neuronal responses may combine with population activity to encode specific social information in both sexes. In this review, we summarize recent findings on how sex-specific sensory information is processed in social behavioral circuits., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In many mammalian species, the accessory olfactory system plays a central role in guiding behavioral and physiological responses to social and reproductive interactions. Because of its relatively compact structure and its direct access to amygdalar and hypothalamic nuclei, the accessory olfactory pathway provides an ideal system to study sensory control of complex mammalian behavior. During the last several years, many studies employing molecular, behavioral, and physiological approaches have significantly expanded and enhanced our understanding of this system. The purpose of the current review is to integrate older and newer studies to present an updated and comprehensive picture of vomeronasal signaling and coding with an emphasis on early accessory olfactory system processing stages. These include vomeronasal sensory neurons in the vomeronasal organ, and the circuitry of the accessory olfactory bulb. Because the overwhelming majority of studies on accessory olfactory system function employ rodents, this review is largely focused on this phylogenetic order, and on mice in particular. Taken together, the emerging view from both older literature and more recent studies is that the molecular, cellular, and circuit properties of chemosensory signaling along the accessory olfactory pathway are in many ways unique. Yet, it has also become evident that, like the main olfactory system, the accessory olfactory system also has the capacity for adaptive learning, experience, and state-dependent plasticity. In addition to describing what is currently known about accessory olfactory system function and physiology, we highlight what we believe are important gaps in our knowledge, which thus define exciting directions for future investigation.

Sensory adaptation is a source of experience-dependent feedback that impacts responses to environmental cues. In the mammalian main olfactory system (MOS), adaptation influences sensory coding at its earliest processing stages. Sensory adaptation in the accessory olfactory system (AOS) remains incompletely explored, leaving many aspects of the phenomenon unclear. We investigated sensory adaptation in vomeronasal sensory neurons (VSNs) using a combination of in situ Ca 2+ imaging and electrophysiology. Parallel studies revealed prominent short-term sensory adaptation in VSNs upon repeated stimulation with mouse urine and monomolecular bile acid ligands at interstimulus intervals (ISIs) less than 30 s. In such conditions, Ca 2+ signals and spike rates were often reduced by more than 50%, leading to dramatically reduced chemosensory sensitivity. Short-term adaptation was reversible over the course of minutes. Population Ca 2+ imaging experiments revealed the presence of a slower form of VSN adaptation that accumulated over dozens of stimulus presentations delivered over tens of minutes. Most VSNs showed strong adaptation, but in a substantial VSN subpopulation adaptation was diminished or absent. Investigation of same- and opposite-sex urine responses in male and female VSNs revealed that adaptation to same-sex cues occurred at ISIs up to 180 s, conditions that did not induce adaptation to opposite-sex cues. This result suggests that VSN sensory adaptation can be modulated by sensory experience. These studies comprehensively establish the presence of VSN sensory adaptation and provide a foundation for future inquiries into the molecular and cellular mechanisms of this phenomenon and its impact on mammalian behavior.

Ca 2+ -activated Cl - currents have been observed in many physiological processes, including sensory transduction in mammalian olfaction. The olfactory vomeronasal (or Jacobson's) organ (VNO) detects molecular cues originating from animals of the same species or from predators. It then triggers innate behaviors such as aggression, mating, or flight. In the VNO, Ca 2+ -activated Cl - channels (CaCCs) are thought to amplify the initial pheromone-evoked receptor potential by mediating a depolarizing Cl - efflux. Here, we confirmed the co-localization of the Ca 2+ -activated Cl - channels anoctamin 1 (Ano1, also called TMEM16A) and Ano2 (TMEM16B) in microvilli of apically and basally located vomeronasal sensory neurons (VSNs) and their absence in supporting cells of the VNO. Both channels were expressed as functional isoforms capable of giving rise to Ca 2+ -activated Cl - currents. Although these currents persisted in the VNOs of mice lacking Ano2 , they were undetectable in olfactory neuron-specific Ano1 knockout mice irrespective of the presence of Ano2 The loss of Ca 2+ -activated Cl - currents resulted in diminished spontaneous and drastically reduced pheromone-evoked spiking of VSNs. Although this indicated an important role of anoctamin channels in VNO signal amplification, the lack of this amplification did not alter VNO-dependent male-male territorial aggression in olfactory Ano1 / Ano2 double knockout mice. We conclude that Ano1 mediates the bulk of Ca 2+ -activated Cl - currents in the VNO and that Ano2 plays only a minor role. Furthermore, vomeronasal signal amplification by CaCCs appears to be dispensable for the detection of male-specific pheromones and for near-normal aggressive behavior in mice., (© 2018 Münch et al.)

The vomeronasal system (VNS) is a major vertebrate chemosensory system that functions in parallel to the main olfactory system (MOS). Despite many similarities, the two systems dramatically differ in the temporal domain. While MOS responses are governed by breathing and follow a subsecond temporal scale, VNS responses are uncoupled from breathing and evolve over seconds. This suggests that the contribution of response dynamics to stimulus information will differ between these systems. While temporal dynamics in the MOS are widely investigated, similar analyses in the accessory olfactory bulb (AOB) are lacking. Here, we have addressed this issue using controlled stimulus delivery to the vomeronasal organ of male and female mice. We first analyzed the temporal properties of AOB projection neurons and demonstrated that neurons display prolonged, variable, and neuron-specific characteristics. We then analyzed various decoding schemes using AOB population responses. We showed that compared with the simplest scheme (i.e., integration of spike counts over the entire response period), the division of this period into smaller temporal bins actually yields poorer decoding accuracy. However, optimal classification accuracy can be achieved well before the end of the response period by integrating spike counts within temporally defined windows. Since VNS stimulus uptake is variable, we analyzed decoding using limited information about stimulus uptake time, and showed that with enough neurons, such time-invariant decoding is feasible. Finally, we conducted simulations that demonstrated that, unlike the main olfactory bulb, the temporal features of AOB neurons disfavor decoding with high temporal accuracy, and, rather, support decoding without precise knowledge of stimulus uptake time. SIGNIFICANCE STATEMENT A key goal in sensory system research is to identify which metrics of neuronal activity are relevant for decoding stimulus features. Here, we describe the first systematic analysis of temporal coding in the vomeronasal system (VNS), a chemosensory system devoted to socially relevant cues. Compared with the main olfactory system, timescales of VNS function are inherently slower and variable. Using various analyses of real and simulated data, we show that the consideration of response times relative to stimulus uptake can aid the decoding of stimulus information from neuronal activity. However, response properties of accessory olfactory bulb neurons favor decoding schemes that do not rely on the precise timing of stimulus uptake. Such schemes are consistent with the variable nature of VNS stimulus uptake., (Copyright © 2018 the authors 0270-6474/18/384957-20$15.00/0.)

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Sexual preference for the opposite sex is a fundamental behavior underlying reproductive success, but the neural mechanisms remain unclear. Here, we examined the role of dopamine signaling in the nucleus accumbens core (NAcc) in governing chemosensory-mediated preference for females in TrpC2 -/- and wild-type male mice. TrpC2 -/- males, deficient in VNO-mediated signaling, do not display mating or olfactory preference toward females. We found that, during social interaction with females, TrpC2 -/- males do not show increased NAcc dopamine levels, observed in wild-type males. Optogenetic stimulation of VTA-NAcc dopaminergic neurons in TrpC2 -/- males during exposure to a female promoted preference response to female pheromones and elevated copulatory behavior toward females. Additionally, we found that signaling through the D1 receptor in the NAcc is necessary for the olfactory preference for female-soiled bedding. Our study establishes a critical role for the mesolimbic dopaminergic system in governing pheromone-mediated responses and mate choice in male mice., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The Bruce effect refers to pregnancy termination in recently pregnant female rodents upon exposure to unfamiliar males [1]. This event occurs in specific combinations of laboratory mouse strains via the vomeronasal system [2, 3]; however, the responsible chemosensory signals have not been fully identified. Here we demonstrate that the male pheromone exocrine gland-secreting peptide 1 (ESP1) is one of the key factors that causes pregnancy block. Female mice exhibited high pregnancy failure rates upon encountering males that secreted different levels of ESP1 compared to the mated male. The effect was not observed in mice that lacked the ESP1 receptor, V2Rp5, which is expressed in vomeronasal sensory neurons. Prolactin surges in the blood after mating, which are essential for maintaining luteal function, were suppressed by ESP1 exposure, suggesting that a neuroendocrine mechanism underlies ESP1-mediated pregnancy failure. The single peptide pheromone ESP1 conveys not only maleness to promote female receptivity but also the males' characteristics to facilitate memorization of the mating partner., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Signal transduction in sensory neurons of the mammalian vomeronasal organ (VNO) involves the opening of the canonical transient receptor potential channel Trpc2, a Ca 2+ -permeable cation channel that is activated by diacylglycerol and inhibited by Ca 2+ -calmodulin. There has been a long-standing debate about the extent to which the second messenger inositol 1,4,5-trisphosphate (InsP 3 ) and type 3 InsP 3 receptor (InsP 3 R3) are involved in the opening of Trpc2 channels and in sensory activation of the VNO. To address this question, we investigated VNO function of mice carrying a knockout mutation in the Itpr3 locus causing a loss of InsP 3 R3. We established a new method to monitor Ca 2+ in the endoplasmic reticulum of vomeronasal sensory neurons (VSNs) by employing the GFP-aequorin protein sensor erGAP2. We also performed simultaneous InsP 3 photorelease and Ca 2+ monitoring experiments, and analysed Ca 2+ dynamics, sensory currents, and action potential or field potential responses in InsP 3 R3-deficient VSNs. Disruption of Itpr3 abolished or minimized the Ca 2+ transients evoked by photoactivated InsP 3 , but there was virtually no effect on sensory activation of VSNs. Therefore, InsP 3 R3 is dispensable for primary chemoelectrical transduction in mouse VNO. We conclude that InsP 3 R3 is not required for gating of Trpc2 in VSNs.

Chemosensory processing in mammals involves the olfactory and vomeronasal systems, but how the activity of both circuits is integrated is unknown. In our study, we recorded the electrophysiological activity in the olfactory bulbs and the vomeronasal amygdala in freely behaving mice exploring a battery of neutral and conspecific stimuli. The exploration of stimuli, including a neutral stimulus, induced synchronic activity in the olfactory bulbs characterized by a dominant theta rhythmicity, with specific theta-gamma coupling, distinguishing between vomeronasal and olfactory structures. The correlated activation of the bulbs suggests a coupling between the stimuli internalization in the nasal cavity and the vomeronasal pumping. In the amygdala, male stimuli are preferentially processed in the medial nucleus, whereas female cues induced a differential response in the posteromedial cortical amygdala. Thus, particular theta-gamma patterns in the olfactory network modulates the integration of chemosensory information in the amygdala, allowing the selection of an appropriate behaviour.

Background: Achieving controlled stimulus delivery is a major challenge in the physiological analysis of the vomeronasal system (VNS)., New Method: We provide a comprehensive description of a setup allowing controlled stimulus delivery into the vomeronasal organ (VNO) of anesthetized mice. VNO suction is achieved via electrical stimulation of the sympathetic nerve trunk (SNT) using cuff electrodes, followed by flushing of the nasal cavity. Successful application of this methodology depends on several aspects including the surgical preparation, fabrication of cuff electrodes, experimental setup modifications, and the stimulus delivery and flushing. Here, we describe all these aspects in sufficient detail to allow other researchers to readily adopt it. We also present a custom written MATLAB based software with a graphical user interface that controls all aspects of the actual experiment, including trial sequencing, hardware control, and data logging., Results: The method allows measurement of stimulus evoked sensory responses in brain regions that receive vomeronasal inputs. An experienced investigator can complete the entire surgical procedure within thirty minutes., Comparison With Existing Methods: This is the only approach that allows repeated and controlled stimulus delivery to the intact VNO, employing the natural mode of stimulus uptake. The approach is economical with respect to stimuli, requiring stimulus volumes as low as 1-2μl., Conclusions: This comprehensive description will allow other investigators to adapt this setup to their own experimental needs and can thus promote our physiological understanding of this fascinating chemosensory system. With minor changes it can also be adapted for other rodent species., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Comparative methods are often used to infer loss or gain of complex phenotypes, but few studies take advantage of genes tightly linked with complex traits to test for shifts in the strength of selection. In mammals, vomerolfaction detects chemical cues mediating many social and reproductive behaviors and is highly conserved, but all bats exhibit degraded vomeronasal structures with the exception of two families (Phyllostomidae and Miniopteridae). These families either regained vomerolfaction after ancestral loss, or there were many independent losses after diversification from an ancestor with functional vomerolfaction. In this study, we use the Transient receptor potential cation channel 2 (Trpc2) as a molecular marker for testing the evolutionary mechanisms of loss and gain of the mammalian vomeronasal system. We sequenced Trpc2 exon 2 in over 100 bat species across 17 of 20 chiropteran families. Most families showed independent pseudogenizing mutations in Trpc2, but the reading frame was highly conserved in phyllostomids and miniopterids. Phylogeny-based simulations suggest loss of function occurred after bat families diverged, and purifying selection in two families has persisted since bats shared a common ancestor. As most bats still display pheromone-mediated behavior, they might detect pheromones through the main olfactory system without using the Trpc2 signaling mechanism., (© 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.)

The aim of this study was to perform a pioneering investigation into the incidence of pneumatization in human skulls. A total of 93 human skulls (≥20 years of age, 69 males, 24 females) were included in the study. The skulls were scanned in a fixed position using cone beam computed tomography (CBCT). The pneumatized space parameters within the nasal septum-width, length, and height-were measured. Two types of finding were identified: (a) Pneumatization, named "sinus septi nasi" (SSN), and (b) "spongy bone" (SB). The results showed SSN in 32 of the 93 skulls (34.4%). The SSN formations were from 0.5 to 4.2 mm wide, 3.5 to 18.8 mm long, and 3.8 to 17.7 mm high. Tumefactions filled with SB were found in 61 of the 93 skulls (65.59%). These were not suitable for precise measurements since the outer borders were not strictly and well defined on CT scans (perhaps because of the preparation process). In conclusion, the perpendicular plate of the ethmoidal bone is not always compact bone; in 34.4% of cases, it shows a degree of pneumatization. In contrast, an enlarged formation filled with SB is present in 65.59% of cases. The possible sources of pneumatization of this little-investigated region are discussed: sphenoid sinus, frontal sinus, and vomeronasal organ. Clin. Anat. 30:312-317, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Surgical or genetic disruption of vomeronasal organ (VNO)-accessory olfactory bulb (AOB) function previously eliminated the ability of male mice to processes pheromones that elicit territorial behavior and aggression. By contrast, neither disruption significantly affected mating behaviors, although VNO lesions reduced males' investigation of nonvolatile female pheromones. We explored the contribution of VNO-AOB pheromonal processing to male courtship using optogenetic activation of AOB projections to the forebrain. Protocadherin-Cre male transgenic mice received bilateral AOB infections with channelrhodopsin2 (ChR2) viral vectors, and an optical fiber was implanted above the AOB. In olfactory choice tests, males preferred estrous female urine (EFU) over water; however, this preference was eliminated when diluted (5%) EFU was substituted for 100% EFU. Optogenetic AOB activation concurrent with nasal contact significantly augmented males' investigation compared to 5% EFU alone. Conversely, concurrent optogenetic AOB activation significantly reduced males' nasal investigation of diluted urine from gonadally intact males (5% IMU) compared to 5% IMU alone. These divergent effects of AOB optogenetic activation were lost when males were prevented from making direct nasal contact. Optogenetic AOB stimulation also failed to augment males' nasal investigation of deionized water or of food odors. Finally, during mating tests, optogenetic AOB stimulation delivered for 30 s when the male was in physical contact with an estrous female significantly facilitated the occurrence of penile intromission. Our results suggest that VNO-AOB signaling differentially modifies males' motivation to seek out female vs male urinary pheromones while augmenting males' sexual arousal leading to intromission and improved reproductive performance.

Pheromones were identified as chemical signals used for intraspecific communication in insects (e.g., sexual attraction) in the 1950s. However, only almost 40 years later the vomeronasal receptors type-1 (V1R) and type-2 (V2R) were identified, usually associated with the presence of a vomeronasal organ (VNO). VRs are widespread in amphibians, reptiles, and mammals, but birds lost the VNO. Similarly, fishes lack VRs and a VNO but can still detect pheromones, instead using the olfactory receptors related to class A and class C G protein-coupled receptors. Here, we review recent evidence on VR repertoire contraction/expansion in vertebrates. We assess the association between VNO development and VR repertoire size. Phylogenetic relationships and selective pressures illuminate the dynamic evolutionary history of the VRs in vertebrates.

This review deals with the structure and function of the vomeronasal system and evaluation of its influence on the sexual sphere of humans and animals. Special attention is given to the role of pheromones in the regulation of the sexual behaviour. The data concerning the function of the vomeronasal organ following surgical interventions in the nasal cavity are discussed.

"Olfactory subsystems" is a relatively new terminology to refer to the different regions of the nasal cavity featuring olfactory sensory neurons. In mice, the olfactory chemical cues are detected in four well delimited areas: the main olfactory epithelium, the septal organ, Grüneberg's ganglion, and the sensory epithelium of the vomeronasal organ. Nevertheless, such distribution is by no means exhibited by all mammals. In microsmatic mammals -humans included- the only existing olfactory subsystem is the main olfactory epithelium. This raises the question of whether the lack of certain olfactory structures in those species implies that they are unable to identify certain olfactory signals, or on the contrary, their main olfactory epithelium assumes such role. It would be interesting to determine, in the context of biomedical research, if the sense of smell in humans is fully or partially endowed with the wide range of functions assigned to the vomeronasal system in mice. If it is not, presumptive implications of the lack of such functions should be addressed in human health and well-being. Anat Rec, 299:1488-1491, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs. The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Different individuals exhibit distinct behaviors, but studying the neuronal basis of individuality is a daunting challenge. Here, we considered this question in the vomeronasal organ, a pheromone-detecting epithelium containing hundreds of distinct neuronal types. Using light-sheet microscopy, we characterized in each animal the abundance of 17 physiologically defined types, altogether recording from half a million sensory neurons. Inter-animal differences were much larger than predicted by chance, and different physiological cell types showed distinct patterns of variability. One neuronal type was present in males and nearly absent in females. Surprisingly, this apparent sexual dimorphism was generated by plasticity, as exposure to female scents or single ligands led to both the elimination of this cell type and alterations in olfactory behavior. That an all-or-none apparent sex difference in neuronal types is controlled by experience-even in a sensory system devoted to "innate" behaviors-highlights the extraordinary role of "nurture" in neural individuality., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A single sensory cue can evoke different behaviors that vary by recipient. Responses may be influenced by sex, internal state, experience, genotype, and coincident environmental stimuli. Pheromones are powerful inducers of mouse behavior, yet pheromone responses are not always stereotyped. For example, male and female mice respond differently to sex pheromones while mothers and virgin females respond differently to pup cues. Here, we review the origins of variability in responses to reproductive pheromones. Recent advances have indicated how response variability may arise through modulation at different levels of pheromone-processing circuitry, from sensory neurons in the periphery to central neurons in the vomeronasal amygdala. Understanding mechanisms underlying conditional pheromone responses should reveal how neural circuits can be flexibly sculpted to alter behavior., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The olfactory and vomeronasal systems of vertebrates are characterised by neurogenesis occurring throughout life. The regenerative ability of olfactory receptor neurons relies on specific glial cells, the olfactory and vomeronasal axon-surrounding cells. Numerous studies have examined mammalian olfactory ensheathing cells which are considered potential candidates for spinal cord injury repair using cell-based therapy. With regard to non-mammalian vertebrates, limited information is available on these glial cells in fish, and there is no information on them in terrestrial anamniotes, the amphibians. In the present research, we studied the immunocytochemical characteristics of axon-surrounding cells in Ambystoma mexicanum. Urodeles have relatively simple olfactory and vomeronasal systems, and represent a good model for studying ensheathing cells in extant representatives of basal tetrapods. Sections from the decalcified heads of A. mexicanum were immunocytochemically processed for the detection of proteins used in research on mammalian olfactory-ensheathing cells. S100, GFAP and NCAM were clearly observed. p75NTR, Gal-1 and PSA-NCAM showed weak staining. No vimentin immunopositivity was observed. The corresponding areas of the olfactory and vomeronasal pathways displayed the same staining characteristics, with the exception of Gal-1, p75NTR and PSA-NCAM in the mucosae. The degree of marker expression was not uniform throughout the sensory pathways. In contrast to fish, both olfactory and vomeronasal nerves displayed uniform staining intensity. This study showed that some markers for mammalian and fish-ensheathing glia are also applicable in urodeles. The olfactory systems of vertebrates show similarities, and also clear dissimilarities. Further investigations are required to ascertain the functional significance of these regional and interspecific differences.

The medial amygdaloid nucleus (Me) is a key node in the socio-sexual brain, composed of anterior (MeA), posteroventral (MePV) and posterodorsal (MePD) subdivisions. These subdivisions have been suggested to play a different role in reproductive and defensive behaviours. In the present work we analyse the afferents of the three Me subdivisions using restricted injections of fluorogold in female outbred CD1 mice. The results reveal that the MeA, MePV and MePD share a common pattern of afferents, with some differences in the density of retrograde labelling in several nuclei. Common afferents to Me subdivisions include: the accessory olfactory bulbs, piriform cortex and endopiriform nucleus, chemosensory amygdala (receiving direct inputs from the olfactory bulbs), posterior part of the medial bed nucleus of the stria terminalis (BSTM), CA1 in the ventral hippocampus and posterior intralaminar thalamus. Minor projections originate from the basolateral amygdala and amygdalo-hippocampal area, septum, ventral striatum, several allocortical and periallocortical areas, claustrum, several hypothalamic structures, raphe and parabrachial complex. MeA and MePV share minor inputs from the frontal cortex (medial orbital, prelimbic, infralimbic and dorsal peduncular cortices), but differ in the lack of main olfactory projections to the MePV. By contrast, the MePD receives preferential projections from the rostral accessory olfactory bulb, the posteromedial BSTM and the ventral premammillary nucleus. In summary, the common pattern of afferents to the Me subdivisions and their interconnections suggest that they play cooperative instead of differential roles in the various behaviours (e.g., sociosexual, defensive) in which the Me has been shown to be involved.